Efficient isolation of Pseudomonas aeruginosa type III secretion translocators and assembly of heteromeric transmembrane pores in model membranes.
Efficient isolation of Pseudomonas aeruginosa type III secretion translocators and assembly of heteromeric transmembrane pores in model membranes.
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有效分离铜绿假单胞菌 III 型分泌易位子并组装模型膜中的异聚跨膜孔。
DOI：
10.1021/bi200905x
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发表时间：
2011
期刊：
影响因子：
2.9
通讯作者：
Heuck,AlejandroP
中科院分区：
文献类型：
--
作者：
Romano,FabianB;Rossi,KyleC;Savva,ChristosG;Holzenburg,Andreas;Clerico,EugeniaM;Heuck,AlejandroP
铜绿假单胞菌（Pseudomonasaerodiosa）通过T3 S系统将细菌毒素或效应物转运到宿主细胞内，是许多革兰氏阴性菌共有的保守机制。蛋白质易位可能通过由两个T3 S分泌的蛋白质易位子PopB和PopD形成的蛋白质跨膜孔发生。未折叠的转位子在插入靶膜之前通过T3 S针分泌。纯化的PopB和PopD在模型膜中形成孔。然而，它们在溶液中形成异质聚集体的倾向阻碍了对这些蛋白质如何从变性状态转变为膜插入状态的分析。将易位体纯化为与同源分子伴侣PcrH的稳定复合物，并使用6 M尿素从分子伴侣中分离。我们在这里报告的组件稳定的跨膜孔的尿素变性的translocators在膜的存在下，通过稀释。PopB和PopD自发结合脂质体含有阴离子磷脂和胆固醇在pH值依赖性的方式观察到的两个独立的测定，时间分辨Fo Erster共振能量转移和蔗糖阶梯梯度超离心。使用Bodipy标记的蛋白质，我们发现PopB与PopD在膜表面上相互作用，通过激发能迁移和荧光猝灭来确定。稳定的跨膜孔在pH <5.0时更有效地组装，表明酸性残基可能参与初始膜结合和/或插入。总之，这里描述的实验装置代表了一种有效的方法，用于重组和分析膜插入的易位。
Translocation of bacterial toxins or effectors into host cells using the type III secretion (T3S) system is a conserved mechanism shared by many Gram-negative pathogens.Pseudomonas aeruginosainjects different proteins across the plasma membrane of target cells, altering the normal metabolism of the host. Protein translocation presumably occurs through a proteinaceous transmembrane pore formed by two T3S secreted protein translocators, PopB and PopD. Unfolded translocators are secreted through the T3S needle prior to insertion into the target membrane. Purified PopB and PopD form pores in model membranes. However, their tendency to form heterogeneous aggregates in solution had hampered the analysis of how these proteins undergo the transition from a denatured state to a membrane-inserted state. Translocators were purified as stable complexes with the cognate chaperone PcrH and isolated from the chaperone using 6 M urea. We report here the assembly of stable transmembrane pores by dilution of urea-denatured translocators in the presence of membranes. PopB and PopD spontaneously bound liposomes containing anionic phospholipids and cholesterol in a pH-dependent manner as observed by two independent assays, time-resolved Förster resonance energy transfer and sucrose-step gradient ultracentrifugation. Using Bodipy-labeled proteins, we found that PopB interacts with PopD on the membrane surface as determined by excitation energy migration and fluorescence quenching. Stable transmembrane pores are more efficiently assembled at pH <5.0, suggesting that acidic residues might be involved in the initial membrane binding and/or insertion. Altogether, the experimental setup described here represents an efficient method for the reconstitution and analysis of membrane-inserted translocators.